Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 199

public sunflower map. An RGC of the TIR-NBS-LRR subclass, originally
named Ha-4W2 and later renamed HaRGC1 , was linked to Pl 1 on LG 8 of the
public map (Gedil et al. 2001a). Mapping of resistance gene candidates
produced by degenerate primers pointed to the clustering of disease resistance
genes in the Pl 1 - Pl 2 - Pl 6 region (Vear et al. 1997).
Using degenerate primers (Leister et al. 1996) and specific primers derived
from sequences of sunflower RGAs (Genbank Accession No. AF272766,
AF272767, AF272768 and AF272769) amplification products were obtained by
PCR using sunflower DNA from the parents of the investigated crosses and two
bulks of the OC YSO cross (Radwan et al. 2003). Sixteen different RGAs were

identified and based on sequence comparison and Southern hybridization
patterns grouped into six classes. Two of these classes correspond to TIR-NBS-
LRR sequences while the remaining four classes correspond to non-TIR-NBS-
LRR type resistance genes. Genetic mapping of these RGAs on two segregating F 2
populations showed that the non-TIR-NBS-LRR RGAs are clustered and linked
to the Pl 5 / Pl 8 locus for resistance to downy mildew in sunflower (Radwan et al.
2003). Radwan et al. (2004) cloned full-length cDNA and genomic sequences
from two of these RGAs, which were named as Ha-NTIR11g (accession no.
AY490793) and Ha-NTIR3a (accession no. AY490791). The genomic sequence
length of Ha-NTIR1g1 was 6,780 bp corresponding to a cDNA of 5,154 bp. This
sequence contains one ORF of 3,848 bp. For Ha-NTIR3 two different clones of
4,034 bp and 3,986 bp were identified with a putative stop codon at position
3,986 for Ha-NTIR3A and 3,861 for Ha-NTIR3B . The predicted protein structures
of the RGA clones Ha-NTIR11g and Ha-NTIR3A determined using the PFAM
( http://pfam.wustl.edu ) and SMART ( http://smart.embl-heidelberg.de ) databases were
1,279 and 1,302 amino acids long, respectively, and showed domains of the
resistance genes of the CC-NBS-LRR class. The genetically incompatible
combination involving the downy mildew race 300 and sunflower line QIR8
carrying the Pl 8 resistance gene is characterized by a hypersensitive-like reaction
(Radwan et al. 2005). Semi-quantitative RT-PCR analysis showed that the
transcript of Ha-NTIR11g was specifically induced during the incompatible
reaction (Radwan et al. 2005). The high level of transcriptional expression of this
RGA coincided with the transcript accumulation of the hsr203J gene, which
is a marker of the hypersensitive reaction. However, treatment with salicylic
acid and methyl jasmonate did not activate transcription of the Ha-NTIR11g
gene, indicating that Ha-NTIR11g is not regulated by defense signaling
pathways triggered by these molecules. In conclusion, a sunflower RGA
Ha-NTIR11g was isolated that is transcriptionally activated in the
incompatible reaction with downy mildew race 300. However, the role of
this gene in sunflower resistance to P. halstedii needs to be verified by
both transforming a susceptible sunflower line and analyzing its complete
promoter (Radwan et al. 2005). Primers flanking an intron between the TIR-
and NBS encoding region of the HaRGC1 sequence amplify a large RGC